Pharmaceutical composition effective against biofilms

ABSTRACT

The invention provides a compound termed Carolacton having the structure and derivatives thereof for medical use against biofilm formation by bacteria.

The present invention relates to a pharmaceutical composition ormedicament, which is effective against bio films formed by or comprisingbacteria.

The present invention provides a pharmaceutical composition effective inthe reduction or prevention of bacterial bio films, which bio films aregenerated on natural or synthetic surfaces in vitro or in vivo, e.g. onthe surface of teeth in the form of dental plaque, or in infectionswhich are chronic and persistent, e.g. cystic fibrosis associatedpneumonia, or on implant surfaces, e.g. of the surface of stents,artificial joints, heartvalves or vessels.

STATE OF THE ART

Rasmussen et al. (Microbiology 152, 895-904 (2006)) give an overview ofthe mechanism leading to the formation of bacterial bio films, involvingthe coordinated gene expression in accordance with population density,which is termed quorum sensing. For gram-negative bacteria, quorumsensing has been identified to involve regulation by the secretion ofdiffusable signal molecules, e.g. acylhomoserine lactones (AHL) by theAHL synthase lux I homologue genes. Accumulation of the single moleculesup to a certain threshold concentration leads to complexing withreceptor proteins, e.g. the luxR gene product (Waters et al. (Annu Rev.Cell Dev. Biol 21, 319-346 (2005)), which AHL-receptor complex is atranscription activator for specific gene cassettes, e.g., forluciferase in Vibrio fischeri. In addition to the activation oftranscription of a specific gene complex, transcription of the AHLsynthase is activated, leading to a self-activating cycle. Rasmussen etal. describe specific competitive inhibitors for the acylated homoserinelactones as well as a screening assay to identify quorum sensinginhibitors using genetically modified bacteria.

Jefferson (FEMS Microbiology Letters 236, 163-173 (2004)) shows that atleast in Staphylococcus aureus bio film formation, the production ofexopoly saccharides is an important factor in the generation andstructure of biofilms.

WO02/099113 A1 describes a compound with the basic structure of formulaI of the present invention, and gives the biological activity as beingantifungal only. Accordingly, there is mentioned the medical use of thecompound as an antifungal agent.

Generally, in clinical infections involving bio film formation, anincreased resistance of bacteria against antibiotics is found.

OBJECTS OF THE INVENTION

The present invention seeks to provide a compound having activity forthe reduction or prevention of bio films, especially of bacterial biofilms. Further, the present invention seeks to provide a pharmaceuticalcomposition comprising the compound having activity against bio filmformation by bacteria. Accordingly, the present invention seeks toprovide the use of a compound for the production of a pharmaceuticalcomposition for use against bio film formation, e.g. for use in theinhibition, reduction or prevention of biofilm formation by bacteria,including pathogenic bacteria.

GENERAL DESCRIPTION OF THE INVENTION

The invention achieves the above-mentioned objects by providing acompound comprising a structure according to formula I, its use for theproduction of pharmaceutical compositions, especially for medical use inthe reduction, prevention and/or inhibition of biofilms. Firstly, thepresent invention provides a compound comprising a structure accordingto formula I, presently termed Carolacton, which is:

Further, the present invention provides derivatives of the compoundaccording to formula I, wherein C-20, which is the carbon of the methylgroup bound to C-3, is replaced by another carbon containing group R1,and wherein the acid group is esterified, i.e. hydrogen of the hydroxylgroup of C-1 is substituted R2.

In (II), R1 and R2 are preferably independently chosen from hydroxy andC₁ to C₁₂ alkyl, alkylene, aryl, arylalkyl or aromatic groups, e.g.methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-heptyl,n-isoheptyl, n-pentyl- or isopentyl, n-hexyl, isohexyl or a C₇ to C₁₂linear, branched or cyclic, saturated, unsaturated or aromatichydrocarbon. For R1 being methyl and R2 being hydroxyl, compound IIgives Carolacton of formula I. Accordingly, the invention also providesthe use of compounds of formula II for the production of pharmaceuticalcompositions, especially for medical use in the reduction, preventionand/or inhibition of bio films, as well as medical compositions andpharmaceutical formulations containing a Carolacton of formula I orformula II for medical use in the reduction, prevention and/orinhibition of bacterial bio films. As a further derivative ofCarolacton, the compound of formula II can carry an alkoxy group,especially a methoxy, ethoxy, or a C₃- to C₁₂-alkoxy group replacing thehydroxyl group linked to C-17 in formula I or II. Compounds, wherein thehydroxyl group of C-17 in formula I or II is replaced by a C₁- toC₁₂-alkoxy group, including alkoxy groups, wherein the alkyl is selectedfrom the group consisting of n-propyl, isopropyl, n-butyl, isobutyl,n-heptyl, n-isoheptyl, n-pentyl- or isopentyl, n-hexyl, isohexyl or a C₇to C₁₂ linear, branched or cyclic, saturated, unsaturated or aromatichydrocarbon radicals, are also referred to as derivatives of Carolacton.

Further derivatives of the compounds according to formula I and formulaII with for use in the production of pharmaceutical and/or cosmeticcosmetic compositions for use in the reduction, prevention and/orinhibition of bacterial biofilms have one or more of the followingsubstitutions: As described for C-17, the hydroxyl group of C-18 can bereplaced by an alkoxy group as described in relation to the hydroxylgroup of C-17; the double bond connecting C-15 and C-16 and/or thedouble bond connecting C-7 and C-8 can each independently behydrogenated to form saturated bonds, i.e. the double bonds can bereplaced by single bonds, including formal saturation of the carbonatoms by additional hydrogen atoms; and/or the carbonyl group of C-5 canbe converted to a hydroxy group.

Derivatives of Carolacton can be produced by total or partial chemicalsynthesis, and preferably by derivatization of Carolacton that isobtained by fermentation and isolation from the fermentation broth.Derivatization reactions for producing derivatives of the invention fromCarolacton obtained by fermentation of a natural producer strain areknown to the skilled person.

In summary, the following compounds are provided having activity againstbacterial bio films, e.g. as components in pharmaceutical compositionsfor medical use and/or in cosmetic compositions:

wherein each of R1, R2, R3 and R4 independently is selected from thegroup comprising or consisting of hydrogen and C_(i)- to C_(u)-alkylgroups, including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,n-heptyl, n-isoheptyl, n-pentyl- or isopentyl, n-hexyl, isohexyl and C₇to C₁₂ linear, branched or cyclic, saturated, unsaturated or aromatichydrocarbon radicals,wherein the bond connecting C-15 and C-16 is a double bond, oralternatively the bond is reduced to a single bond with C-15 and C-16being saturated with hydrogen atoms,wherein the bond connecting C-7 and C-8 is a double bond, oralternatively the bond is reduced to a single bond with C-7 and C-8being saturated with hydrogen atoms, and wherein C-5 carries a carbonylgroup, or alternatively is reduced to a hydroxyl group.

Carolacton has no relevant antibiotic activity. Concentrations above 40μg/mL are needed to inhibit E. coli. Against E. coli tolC, which has animpaired cell wall, an antibiotic activity at 60 ng/mL in vitro has beenfound.

Especially Carolacton as well as compounds comprising a structureaccording to formula II and III have been found to reduce or inhibit theformation of biofilms at very low concentrations, e.g. exemplified byStreptococcus mutans, a clinically important contributor to pathogenicbio films, e.g. in the generation of caries and endocarditis, preferablyunder anaerobic conditions like on surfaces of implants within the humanor animal body. The inhibition of biofilm generation is achieved atconcentrations as low as 0.005 μg/mL in in vitro cultures.

Compounds comprising a structure according to formulae II and III,exemplified by Carolacton of formula I, do not have a pronounced generalantibiotic effect against bacteria. Therefore, a negative side effectfrom a general antibiotic activity of compositions comprising a compoundaccording to formula II or III, e.g. of Carolacton, is essentiallyavoided.

Preferably, Carolacton or a compound according to formula II oraccording to formula III can be used as the active ingredient inpharmaceutical and in cosmetic compositions having an activity againstbacterial bio film formation, the biofilms including or essentiallyconsisting of Gram-negative and/or Gram-positive bacteria, e.g. thebiofilm comprising Streptococcus pneumoniae, Streptococcus pyogenes,Peptococcus/Peptostreptococcus sp., Staphylococcus aureus,Staphylococcus epidermidis, Burkholderia cepacia, Pseudomonasaeruginosa, Enterococcus faecalis, E. coli, and clinical isolates ofthese, including pathogenic strains.

Further, the invention provides pharmaceutical composition, preferablyfurther comprising non-toxic and pharmaceutically acceptable carrier,diluent, bulking and/or formulating agents, and at least one compound ofthe invention as the active ingredient. In accordance with thepharmaceutical activity of Carolacton and its derivatives, especiallyaccording to formulae I and II including derivatives according toformula III, the pharmaceutical composition of the invention ispreferably provided for the reduction, prevention and/or inhibition of abacterial biofilm, e.g. for the medical indication or medical diagnosisrequiring the reduction, prevention and/or inhibition of a bacterial biofilm, especially of an anaerobic bacterial biofilm. Accordingly, thepharmaceutical composition of the invention can be marked to be activeor suitable for the reduction, prevention and/or inhibition of abacterial bio film in medical indications requiring the reduction,prevention and/or inhibition of a bacterial biofilm. Further, thepharmaceutical composition of the invention can be marked to be activeor suitable only for the reduction, prevention and/or inhibition of abacterial bio film in medical indications requiring the reduction,prevention and/or inhibition of a bacterial bio film. Such medicalindications include but are not limited to bacterial bio films oninternal and external surfaces of hard and soft tissue, includingmucosal surfaces, internal blood vessel surfaces, bone and cartilagesurfaces, tooth surfaces, and the eye, especially in human beings, andbacterial bio films on implants of natural and/or synthetic materials,which implants can be arranged within a human body.

Further, the invention provides cosmetic compositions including at leastone Carolacton compound, and the use of compositions containing at leastone Carolacton compound for cosmetic purposes, e.g. a dental careformulation, compositions for use in the prevention or reduction ofbacterial infections in the human eye, including contact lens careformulations. Further, the pharmaceutical compositions and the cosmeticcompositions can be for use in indications requiring the reduction,prevention and/or inhibition of a bacterial bio film under essentiallyanaerobic conditions, and the compositions can be marked to be suitablefor that use or indication, or marked to be suitable for that use orindication only.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is now described in greater detail with referenceto the FIGURE, showing a graphic representation of biofilm inhibition inresponse to increasing concentrations of Carolacton under in vitroconditions.

Example 1 Production of Compounds Comprising a Structure According toFormula I (Carolacton) and its Derivatives

Compounds comprising a structure according to formula II can be producedfrom Carolacton according to formula I by derivatization, e.g. bytransesterification to substitute R1 and/or R2 by different radicals,e.g. involving the use of standard synthesis methods, e.g. involving thebinding and removal of protecting groups to atoms of formula I.

Carolacton can be produced by fermentation of myxobacteria, especiallyof Sorangium cellulosum, also called Polyangium cellulosum. Forfermentation, strain DSM 19571, available from DSMZ GmbH, Braunschweig,Germany, was cultivated at 30° C. under aerobic conditions on agarplates containing casitone, Difco, 0.3%; CaCl₂×2H₂O, 0.1%; yeastextract, Difco, 0.1%; agar 1.5% at pH 7.2, including a carbon source,e.g. glucose or starch at 0.1%. Alternatively, agar is used, containing0.5% fresh baker's yeast and 0.1 calcium chloride at 1.5% agar, pH 7.2.

For fermentation, peptone from tryptically digested casein, 0.3%,calcium chloride at 0.05%, magnesium chloride at 0.2% and a carbonsource, glucose or starch, at 0.1% can be used. Preferably, thefollowing liquid medium is used for production of Carolacton: 0.8%starch, 0.2% yeast extract, 0.2% soy meal with fat removed, 0.1% calciumchloride, 1% magnesium chloride, 0.2% glucose, 8 mg/L NaFe(III)-EDTA,1.19% HEPES-buffer, pH adjusted to 7.4 prior to autoclaving using 20%potassium hydroxide.

For the pre-culture, the preferred medium was used in 6 parallel2-L-Erlenmeyer flasks containing 800 mL medium, inoculated with 60 mLculture each. The pre-culture was incubated on a rotary shaker at 160rpm for three days at 30° C. For fermentation, the preferred medium wasused, but omitting HEPES, maintaining the pH at or above 0.7 using 5%potassium hydroxide. To the medium, 1.5% (v/v) adsorber resin AmberliteXAD 16 (Rohm and Haas) was added. The stirrer speed was 100 rpm,aeration was at 1.0 v/v min. Carolacton production was measured usingthe antibacterial activity against E. coli tolC by extracting an aliquotfrom the fermenter after removal of XAD resin with methanol, andconcentrating the methanol extract to 1/25 of the initial sample volume.Of the extract, 10 μL were applied to an antibiotic assay disc with 6 mmdiameter and placed onto an agar plate inoculated with the testorganism. After incubation for one day at 30° C., the inhibition zonewas measured. Glucose was determined using the DIABUR—test 5000 teststicks available from Roche. The course of the fermentation is shown intable 1:

TABLE 1 Course of fermentation pO₂ Inhibition zone diameter of Time(days) pH (%) Glucose (%) E. coli tolC (mm)  0 (start)  3 7.06 85 n.d.n.d.  4 7.07 50 0.3 n.d.  5 7.13 39 0.3  7  6 7.16 28 0.25 10  7  7.03.29 0.1 15 10 7.25 28 0 21 11 7.27 28 0 21 n.d. = not determined

The fermentation was stopped on day 11. XAD was removed by sieving,Carolacton was produced to a final concentration of 3.4 mg/L broth.

From 100 L fermentation broth, the adsorber resin was harvested byfiltration (210 μm pore size). The resin was washed with water forremoval of adherent cells and extracted with 30% methanol in water.Elution was done with methanol (8 L) to yield raw Carolacton. Afterevaporation of the methanol, the residual water layer was extractedthree times with ethyl acetate. The organic layer was dried withanhydrous sodium sulfate, filtrated, concentrated in vacuo, redissolvedin methanol and extracted with n-hexane. After partition and removal ofthe hexane layer, methanol was evaporated to give a crude extract of16.6 g.

For further purification, chromatography on a Sephadex LH20 (column 8×79cm, eluent methanol, flow rate at 28 mL/min) was used. Thin layerchromatography and UV detection identified a spot containing a 8.2 gfraction of Carolacton, which was further separated on a Merck Prep bar100 chromatography system (column 10×40 cm, 120A 15 μm; solvent:methanol/ammonia acetate buffer at pH 5, 57:43, flow 17 mL/min, UVdetection at 210 nm). After evaporation of the Carolacton containingfraction (1.3 g), preparative reverse phase chromatography is performed(column 3×48 cm: Kronlab ODS AQ 120A 16 μL, solvent: acetonitrile/0.05 Mammonia acetate buffer adjusted to pH 5, 65:35, flow rate at 17 mL/min,UV detection at 206 nm) to yield 275 mg Carolacton after evaporation.

Carolacton of formula I could be characterized as follows: FormulaC₂₅H₄₀O₈, MW=468.6, HRMS: [Cl⁻] calculated: 468.2723, found: 468.2732,IR: ν[cm⁻¹] (1 gε)=204 (4.06), 259 (2.35), 290 (2.58); TLC (silica gel254 nm): ethyl acetate/methanol/water 65/30/10, R_(f)=0.62; HPLC:solvent A (95/5 water/acetonitrile plus 5 mM NH₄Ac, pH 5.5), solvent B(5/95 water/acetonitrile plus 5 mM NH₄Ac, pH 5.5) gradient in 30 minfrom 10% B to 100% B, 10 min isocratic B, column 2×125 mm Nucleosil 1205 μm C18 (Macherey Nagel), flow 0.3 mL/min, Rt=12.8 min.

For synthesis of derivatives of Carolacton compounds according toformula II and of formula III, the Carolacton obtained by fermentationwas derivatized, e.g. by alkylating and hydrogenating reagents.Optionally, protective groups were introduced for regio-selectivederivatization. Derivatives could be isolated from derivatizationreaction compositions by standard procedures, preferably by HPLC.

¹H- and ¹³C-NMR data (600/150 MHz) of Carolacton (I) in dichloroform H δm J (Hz) C δ  2 H_(b) 2.64 dd 15.5, 4.2 1 175.6  2 H_(a) 2.43 dd 15.7,5.9 2 35.9  3 H 3.73 ddd 8.5, 5.5, 4.5 3 80.1  4 H 2.97 dq 8.3, 7.2 447.3  6 H 3.48 dq 10.2, 6.8 5 213.3  7 H 5.36 d 10.2  6 47.5  9 H 4.74 d11.3  7 129.4 10 H 2.04 m — 8 135.8 11 H_(b) 1.75 m — 9 83.3 11 H_(a)0.99 m — 10 33.4 12 H_(b) 1.25 m — 11 28.4 12 H_(a) 0.99 m — 12 18.9 13H_(b) 1.37 m — 13 34.4 13 H_(a) 1.25 m — 14 35.7 14 H 2.31 m — 15 134.515 H 5.44 ddd 15.1, 9.8, 16 125.5 1.5 16 H 5.52 dd 15.5, 2.3 17 73.0 17H 4.47 s br. 18 73.7 18 H 4.16 d 3.8 19 172.0 20 H₃ 3.30 s — 20 58.1 21H₃ 0.92 d 6.8 21 12.8 22 H₃ 1.10 d 6.8 22 15.3* 23 H₃ 1.70 s — 23 13.024 H₃ 0.76 d 7.2 24 15.3* 25 H₃ 0.97 d 6.4 25 21.7 *interchangeable

Example 2 Inhibition of Bio Film Formation in In Vitro Culture

Using Streptococcus mutans as a model organism that generates biofilms,overnight cultures of Streptococcus mutans in THB medium (Todd HewittBroth, available from Bacto) were diluted 1:100 into fresh THB mediumcontaining 0.5% wt/vol sucrose. For anaerobic growth, medium was flushedwith nitrogen before use. Aliquots of the diluted culture (95 μL) ofExample 1 were distributed into the wells of a 96-well polystyrene flatbottom microtiter plate, containing 5 μL of different concentrations oftest compound or alternatively, 5 μL methanol as a control. Microtiterplates were incubated under aerobic and an anaerobic conditions,respectively, at 37° C. for approximately 24 hours. For non-biofilmforming growth, e.g. planctonic growth, cells were grown in THB withoutthe additional sucrose under otherwise identical conditions.

Bacterial growth was monitored in the microtiter plate by opticaldensity readings at 620 nm. Quantitative bio film formation wasdetermined using the live/dead bacLight bacterial viability staining kit(available under catalogue number L13152 from Molecular Probes, Eugene,Oreg., USA). The kit incorporates two nucleic acid stains that differ inspectral characteristics and their ability to penetrate bacterial cellmembranes. When used alone, the green fluorescing stain Cyto9 generallylabels all bacteria in the population, whereas the red fluorescing stainpropidium-iodide only penetrates bacteria having damaged membranes,causing a reduction in the Cyto9 stain fluorescence when both dyes arepresent.

For staining, the culture medium was removed from the microtiter platewells and cells were washed once with 100 μL fresh THB medium to removeplanctonic growing and loosely bound cells. According to all themanufacturer's instructions, the adherent bacteria, regarded as the biofilm, were then stained for 15 min in the dark at room temperature,using 100 μL of a 1:1 mixture of the two dye components. Fluorescencewas measured in a microtiter plate reader (Wallac Victor 1420 multilabelcounter, PerkinElmer Life Sciences), equipped with detectors and filtersets for monitoring red and green fluorescence.

Quantitative bio film formation was calculated by dividing thefluorescence intensities of the stained biofilms for each well of themicrotiter plate at an emission of 530 nm (green) by the fluorescenceintensity at the emission of 630 nm (red). As 100% activity, the resultsobtained from wells only containing methanol as the control, instead ofa test compound, was used. Under anaerobic conditions, Carolactoninhibited formation of biofilm from Streptococcus mutans at very lowconcentrations. Results are given in table 3 for Carolacton of formulaI. Planctonic cultures were only slightly influenced at higherconcentrations, as was determined by optical density readings.

TABLE 3 The inhibition of biofilm formation of Streptococcus mutansunder anaerobic and aerobic conditions by Carolacton Carolactonconcentration Biofilm inhibition (%) Biofilm inhibition (%) (μg/mL)under aerobic conditions under anaerobic conditions 50 1 82 25 66 2.5 1960 0.25 21 53; 77 (second experiment) 0.05 60 0.025 21 66 0.005 350.0025 −7

Results for anaerobic bio film inhibition are also shown in the FIGURE,giving a concentration dependent inhibition of biofilm formation in theform of a saturation curve for Carolacton concentrations up to 20 μg/mL.

For a determination of bio film inhibition in relation to the control,the activity value for the Carolacton comprising test well wassubtracted from the value obtained for the control. At concentrations of2.5 and 0.25 μg/mL Carolacton, respectively, bio film formation byStreptococcus mutans wild-type strain was inhibited by about 60% underanaerobic conditions.

When using quorum sensing negative mutants of Streptococcus mutans,which are defective in the quorum sensing pathway, the following resultshave been obtained the same testing conditions:

Streptococcus mutans com E (−) mutant: No significant inhibition; at aconcentration of 2.5 μ/mL, Carolacton, 5% inhibition; at 0.25 μg/mLCarolacton, 12% inhibition. Streptococcus mutans com D (−) mutant: Nosignificant inhibition, namely at both 2.5 μg and 0.25 μg/mL Carolacton,about 10% inhibition. Streptococcus mutans com C (−) mutant: Inductionof biofilm formation between 150% (0.25 μg/mL Carolacton) and 200% (2.5μg/mL Carolacton). Streptococcus mutans luxS mutant: Inhibition similarto wild-type (between 59% (2.5 μg/mL) and 46% (40.25 μg/mL),respectively).

At a concentration of 0.025 μg/mL Carolacton, biofilm formation underanaerobic conditions still was inhibited in vitro by about 65%.

In contrast to growth in bio films, planctonic growth was not influencedby Carolacton, or only marginally reduced. From the results with quorumsensing negative mutants, it can be inferred that Carolacton possiblyinterferes with signalling molecules or signal transduction of thequorum sensing systems.

It could be shown in these examples of Streptococcus mutans thatCarolacton effectively inhibits formation of bacterial bio filmsespecially under anaerobic conditions at low concentrations.Accordingly, a preferred use of the compounds of the invention is forthe production of a pharmaceutical composition for medical or cosmeticuse in conditions where anaerobic conditions dominate, e.g. in cosmeticand/or medical applications against dental plaque formation or inmedical applications against biofilm generation within the human oranimal body, e.g. on implant surfaces.

Similar results indicating activity against bacterial biofilm formationcould be obtained for the Carolacton derivatives of formulae II and III.

1. Use of a compound comprising a structure according to formula III

wherein each of R1, R2, R3 and R4 independently is selected from thegroup comprising hydrogen and C₁- to C₁₂-alkyl groups, including methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-heptyl, n-isoheptyl,n-pentyl- or isopentyl, n-hexyl, isohexyl and C₇ to C₁₂ linear, branchedor cyclic, saturated, unsaturated or aromatic hydrocarbon radicals, forthe production of a pharmaceutical composition for inhibition, reductionor prevention of the generation of bacterial biofilms.
 2. Use accordingto claim 1, wherein the bond connecting C-15 and C-16 is hydrogenated toa single bond and C-15 and C-16 are saturated with hydrogen atoms. 3.Use according to claim 1, wherein the bond connecting C-7 and C-8 ishydrogenated to a single bond and C-7 and C-8 are saturated withhydrogen atoms.
 4. Use according to claim 1, wherein the carbonyl groupof C-5 is reduced to a hydroxyl group.
 5. Use according to claim 1,wherein the compound is according to formula II

wherein R1 and R2 are independently chosen from the group comprising H,hydroxy and C₁ to C₁₂ alkyl, alkylene, aryl, arylalkyl or aromaticgroups, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-heptyl,n-isoheptyl, n-pentyl- or isopentyl, n-hexyl, isohexyl or a C₇ to C₁₂linear, branched or cyclic, saturated, unsaturated or aromatichydrocarbon.
 6. Use according to claim 1, wherein the compound isCarolacton according to formula I


7. Use according to claim 1, characterized in that the bacterial biofilmcomprises pathogenic bacteria.
 8. Use according to claim 1,characterized in that biofilm formation is under essentially anaerobicconditions.
 9. Use according to claim 8, characterized in that theanaerobic conditions are present on surfaces of artificial implants ormedical devices that are arranged within the human or animal body. 10.Use according to claim 1, characterized in that the compound is producedby a process including a fermentation of Sorangium cellulosum and theisolation of Carolacton according to formula I from the fermentationbroth.
 11. Pharmaceutical composition for medical treatment for theprevention of bacterial biofilm formation, the composition comprising asan active ingredient a compound comprising a structure according toformula III

wherein each of R1, R2, R3 and R4 independently is selected from thegroup comprising hydrogen and C₁- to C₁₂-alkyl groups, including methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-heptyl, n-isoheptyl,n-pentyl- or isopentyl, n-hexyl, isohexyl and C₇ to C₁₂ linear, branchedor cyclic, saturated, unsaturated or aromatic hydrocarbon radicals. 12.Pharmaceutical composition according to claim 11, wherein the bondconnecting C-15 and C-16 is hydrogenated to a single bond and C-15 andC-16 are saturated with hydrogen atoms.
 13. Pharmaceutical compositionaccording to claim 11, wherein the bond connecting C-7 and C-8 ishydrogenated to a single bond and C-7 and C-8 are saturated withhydrogen atoms.
 14. Pharmaceutical composition according to claim 11,wherein the carbonyl group of C-5 is reduced to a hydroxyl group. 15.Pharmaceutical composition according to claim 11, wherein the compoundis according to formula II

wherein R1 and R2 are independently chosen from the group comprising H,hydroxy and C₁ to C₁₂ alkyl, alkylene, aryl, arylalkyl or aromaticgroups, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-heptyl,n-isoheptyl, n-pentyl- or isopentyl, n-hexyl, isohexyl or a C₇ to C₁₂linear, branched or cyclic, saturated, unsaturated or aromatichydrocarbon.
 16. Pharmaceutical composition according to claim 11,wherein the compound is Carolacton according to formula I


17. Composition for cosmetic purposes for the prevention, inhibition orreduction of bacterial biofilm formation, the composition comprising asan active ingredient a compound according to formula III

wherein each of R1, R2, R3 and R4 independently is selected from thegroup comprising hydrogen and C₁- to C₁₂-alkyl groups, including methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-heptyl, n-isoheptyl,n-pentyl- or isopentyl, n-hexyl, isohexyl and C₇ to C₁₂ linear, branchedor cyclic, saturated, unsaturated or aromatic hydrocarbon radicals. 18.Composition for cosmetic purposes according to claim 17, wherein thebond connecting C-15 and C-16 is hydrogenated to a single bond and C-15and C-16 are saturated with hydrogen atoms.
 19. Composition for cosmeticpurposes according to claim 17, wherein the bond connecting C-7 and C-8is hydrogenated to a single bond and C-7 and C-8 are saturated withhydrogen atoms.
 20. Composition for cosmetic purposes according to claim17, wherein the carbonyl group of C-5 is reduced to a hydroxyl group.21. Composition for cosmetic purposes according to claim 17, wherein thecompound is according to formula II

wherein R1 and R2 are independently chosen from the group comprising H,hydroxy and C₁ to C₁₂ alkyl, alkylene, aryl, arylalkyl or aromaticgroups, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-heptyl,n-isoheptyl, n-pentyl- or isopentyl, n-hexyl, isohexyl or a C₇ to C₁₂linear, branched or cyclic, saturated, unsaturated or aromatichydrocarbon.
 22. Composition for cosmetic purposes according to claim17, wherein the compound is Carolacton according to formula I


23. Composition according to claim 11, wherein the bacterial biofilmformation is on the surface of a hard tissue or of a soft tissue of thehuman body, or on the surface of an implant having a natural orsynthetic material surface.
 24. Composition according to claim 23,wherein the surface of a hard or soft tissue is selected from the groupconsisting of mucosa, the eye, bone tissue, cartilage tissue, and bloodvessels.